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Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity.

Burbage M, Keppler SJ, Gasparrini F, Martínez-Martín N, Gaya M, Feest C, Domart MC, Brakebusch C, Collinson L, Bruckbauer A, Batista FD - J. Exp. Med. (2014)

Bottom Line: Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization.Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell-intrinsic block in plasma cell differentiation.Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lymphocyte Interaction Laboratory, Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, England, UK.

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Cdc42 is important for early B cell development in the bone marrow. (A) Genetic approach used to ablate Cdc42 specifically in B cells. (B and C) Bone marrow from WT and Cdc42 KO mice was analyzed by flow cytometry. The gating strategies are shown on the left. B cell progenitors were divided into 3 populations on the basis of CD43 and B220 expression levels (B220+CD43+, B220+CD43−, and mature recirculating cells [B220hiCD43−]) as shown in B. They were further subdivided on the basis of CD24, BP-1, IgM, and IgD expression levels into populations A(B220+CD43+CD24−BP-1−), B(B220+CD43+CD24+BP-1−), C(B220+CD43+CD24+BP-1+), D(B220+CD43−IgM−IgD−), E(B220+CD43−IgM+IgD−/Int), and nonrecirculating (IgDhiB220+CD43−IgM+/IntIgD+) as shown in C. Quantifications are shown in the right-hand column and indicate percentage of cells in the indicated gates. Data were pooled from at least 4 independent experiments with at least 2 mice in each group. (D) B cell progenitors (A–E) were sorted from the bone marrow and protein expression of Cdc42 (first row) and actin (second row) were analyzed by Western blot. Densitometric analysis was used to quantify the signal intensity of Cdc42 normalized to actin and is shown in the chart below. (E) In vivo labeling of bone marrow progenitors. Intravenously injected anti CD45.2-PE was used to distinguish cells in BM sinusoid (PE+) from cells in BM parenchyma (PE−). The first two columns show a representative example of one WT mouse (blue) and one Cdc42 KO mouse (red). Populations D, E, and E’ are shown. Quantification charts are shown in the two right-hand columns; data were pooled from three independent experiments with five mice in each group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
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fig1: Cdc42 is important for early B cell development in the bone marrow. (A) Genetic approach used to ablate Cdc42 specifically in B cells. (B and C) Bone marrow from WT and Cdc42 KO mice was analyzed by flow cytometry. The gating strategies are shown on the left. B cell progenitors were divided into 3 populations on the basis of CD43 and B220 expression levels (B220+CD43+, B220+CD43−, and mature recirculating cells [B220hiCD43−]) as shown in B. They were further subdivided on the basis of CD24, BP-1, IgM, and IgD expression levels into populations A(B220+CD43+CD24−BP-1−), B(B220+CD43+CD24+BP-1−), C(B220+CD43+CD24+BP-1+), D(B220+CD43−IgM−IgD−), E(B220+CD43−IgM+IgD−/Int), and nonrecirculating (IgDhiB220+CD43−IgM+/IntIgD+) as shown in C. Quantifications are shown in the right-hand column and indicate percentage of cells in the indicated gates. Data were pooled from at least 4 independent experiments with at least 2 mice in each group. (D) B cell progenitors (A–E) were sorted from the bone marrow and protein expression of Cdc42 (first row) and actin (second row) were analyzed by Western blot. Densitometric analysis was used to quantify the signal intensity of Cdc42 normalized to actin and is shown in the chart below. (E) In vivo labeling of bone marrow progenitors. Intravenously injected anti CD45.2-PE was used to distinguish cells in BM sinusoid (PE+) from cells in BM parenchyma (PE−). The first two columns show a representative example of one WT mouse (blue) and one Cdc42 KO mouse (red). Populations D, E, and E’ are shown. Quantification charts are shown in the two right-hand columns; data were pooled from three independent experiments with five mice in each group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Mentions: In the light of the emerging importance of the cytoskeleton in regulating B cell functions, we sought to characterize the role of the RhoGTPase Cdc42 during B cell development and activation in vivo. To do this, conditionally targeted Cdc42flox/flox mice were crossed with mice expressing Cre recombinase under the promoter of the mb1 gene (Fig. 1 A; Wu et al., 2006; Hobeika et al., 2006). As mb1 encodes the Igα subunit of the BCR, this approach circumvents limitations associated with gene targeting through the Cd19 locus, which is known in some cases to lead to incomplete deletion, especially in the early B cell lineage (Rickert et al., 1997). Indeed, we observed that although Cdc42 was highly expressed early during B cell development in WT controls, expression was completely abrogated in Cdc42flox/floxmb1Cre+/− mice (hereafter designated Cdc42 KO; Fig. 1 D).


Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity.

Burbage M, Keppler SJ, Gasparrini F, Martínez-Martín N, Gaya M, Feest C, Domart MC, Brakebusch C, Collinson L, Bruckbauer A, Batista FD - J. Exp. Med. (2014)

Cdc42 is important for early B cell development in the bone marrow. (A) Genetic approach used to ablate Cdc42 specifically in B cells. (B and C) Bone marrow from WT and Cdc42 KO mice was analyzed by flow cytometry. The gating strategies are shown on the left. B cell progenitors were divided into 3 populations on the basis of CD43 and B220 expression levels (B220+CD43+, B220+CD43−, and mature recirculating cells [B220hiCD43−]) as shown in B. They were further subdivided on the basis of CD24, BP-1, IgM, and IgD expression levels into populations A(B220+CD43+CD24−BP-1−), B(B220+CD43+CD24+BP-1−), C(B220+CD43+CD24+BP-1+), D(B220+CD43−IgM−IgD−), E(B220+CD43−IgM+IgD−/Int), and nonrecirculating (IgDhiB220+CD43−IgM+/IntIgD+) as shown in C. Quantifications are shown in the right-hand column and indicate percentage of cells in the indicated gates. Data were pooled from at least 4 independent experiments with at least 2 mice in each group. (D) B cell progenitors (A–E) were sorted from the bone marrow and protein expression of Cdc42 (first row) and actin (second row) were analyzed by Western blot. Densitometric analysis was used to quantify the signal intensity of Cdc42 normalized to actin and is shown in the chart below. (E) In vivo labeling of bone marrow progenitors. Intravenously injected anti CD45.2-PE was used to distinguish cells in BM sinusoid (PE+) from cells in BM parenchyma (PE−). The first two columns show a representative example of one WT mouse (blue) and one Cdc42 KO mouse (red). Populations D, E, and E’ are shown. Quantification charts are shown in the two right-hand columns; data were pooled from three independent experiments with five mice in each group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4291523&req=5

fig1: Cdc42 is important for early B cell development in the bone marrow. (A) Genetic approach used to ablate Cdc42 specifically in B cells. (B and C) Bone marrow from WT and Cdc42 KO mice was analyzed by flow cytometry. The gating strategies are shown on the left. B cell progenitors were divided into 3 populations on the basis of CD43 and B220 expression levels (B220+CD43+, B220+CD43−, and mature recirculating cells [B220hiCD43−]) as shown in B. They were further subdivided on the basis of CD24, BP-1, IgM, and IgD expression levels into populations A(B220+CD43+CD24−BP-1−), B(B220+CD43+CD24+BP-1−), C(B220+CD43+CD24+BP-1+), D(B220+CD43−IgM−IgD−), E(B220+CD43−IgM+IgD−/Int), and nonrecirculating (IgDhiB220+CD43−IgM+/IntIgD+) as shown in C. Quantifications are shown in the right-hand column and indicate percentage of cells in the indicated gates. Data were pooled from at least 4 independent experiments with at least 2 mice in each group. (D) B cell progenitors (A–E) were sorted from the bone marrow and protein expression of Cdc42 (first row) and actin (second row) were analyzed by Western blot. Densitometric analysis was used to quantify the signal intensity of Cdc42 normalized to actin and is shown in the chart below. (E) In vivo labeling of bone marrow progenitors. Intravenously injected anti CD45.2-PE was used to distinguish cells in BM sinusoid (PE+) from cells in BM parenchyma (PE−). The first two columns show a representative example of one WT mouse (blue) and one Cdc42 KO mouse (red). Populations D, E, and E’ are shown. Quantification charts are shown in the two right-hand columns; data were pooled from three independent experiments with five mice in each group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
Mentions: In the light of the emerging importance of the cytoskeleton in regulating B cell functions, we sought to characterize the role of the RhoGTPase Cdc42 during B cell development and activation in vivo. To do this, conditionally targeted Cdc42flox/flox mice were crossed with mice expressing Cre recombinase under the promoter of the mb1 gene (Fig. 1 A; Wu et al., 2006; Hobeika et al., 2006). As mb1 encodes the Igα subunit of the BCR, this approach circumvents limitations associated with gene targeting through the Cd19 locus, which is known in some cases to lead to incomplete deletion, especially in the early B cell lineage (Rickert et al., 1997). Indeed, we observed that although Cdc42 was highly expressed early during B cell development in WT controls, expression was completely abrogated in Cdc42flox/floxmb1Cre+/− mice (hereafter designated Cdc42 KO; Fig. 1 D).

Bottom Line: Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization.Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell-intrinsic block in plasma cell differentiation.Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lymphocyte Interaction Laboratory, Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, England, UK.

Show MeSH
Related in: MedlinePlus